Method for detecting target nucleic acid

Abstract

A target nucleic acid having a target sequence in a sample is detected according to the steps of: (a) mixing a first probe including a nucleic acid which has a specific region having a sequence complementary to the target sequence and a nonspecific region having a sequence that is not complementary to the target sequence of the target nucleic acid; a second probe including a nucleic acid which has a first region that is complementary to at least a portion of the nonspecific region of the first probe, a loop region that does not have a sequence complementary to the first probe, and a second region that is complementary to at least a portion of the specific region of the first probe, the loop region being capable of forming a loop when it is annealed with the first probe, wherein the nucleic acid is labeled with a labeling material generating a signal by which formation of the aforementioned loop can be detected; and a sample under conditions in which the first probe and the second probe are annealed and the first probe and the target nucleic acid are annealed; and (b) detecting a signal of the labeling material.

1. A method for detecting a target nucleic acid having a target sequence in a sample, comprising the steps of:
 (a) obtaining a first probe comprising a nucleic acid which has a specific region having a sequence complementary to the target sequence and a nonspecific region having a sequence that is not complementary to the target sequence of the target nucleic acid; and a second probe comprising a nucleic acid which has a first region that is complementary to at least a portion of the nonspecific region of the first probe, a loop region that does not have a sequence complementary to the first probe, and a second region that is complementary to at least a portion of the specific region of the first probe, wherein the nucleic acid of the second probe is labeled with a labeling material generating a signal by which formation of a loop can be detected; 
 (b) mixing the first probe and the second probe, thereby forming a mixture wherein the loop in the loop region of the second probe is formed when the second probe is annealed with first probe, thereby quenching the signal from the labeling material in the absence of a target nucleic acid; 
 (c) adding the sample to the mixture of step (b), whereby the target nucleic acid in the sample anneals with the first probe, thereby releasing the second probe; and 
 (d) detecting an increase in the signal of the labeling material in the presence of the target nucleic acid compared to the signal obtained in step (b), thereby detecting the target nucleic acid, wherein the signal is quenched when the first probe and the second probe are annealed and not quenched when the first probe and the second probe are not annealed. 
 
     
     
       2. The method according to  claim 1 , wherein the second region of the second probe is shorter than the specific region of the first probe. 
     
     
       3. The method according to  claim 1 , wherein the labeling material comprises a fluorescent material and a quencher that quenches a fluorescence of the fluorescent material when the quencher is near the fluorescent material, arranged so as to sandwich the loop region, with the fluorescence of the fluorescent material being quenched by the quencher when the first probe and the second probe are annealed to form the loop and not quenched when the first probe and the second probe are not annealed. 
     
     
       4. The method according to  claim 1 , wherein the detection of the increase in the signal is performed quantitatively, thereby quantifying the target nucleic acid.